Process for the Isolation and Stabilization of Low-Molecular Weight Aminoglycans from Waste Egg Shells

ABSTRACT

Processes for the isolation of low-molecular weight aminoglycan compound of formula I from a hitherto unexploited natural source of waste egg shells which is made up of alternating glucuronic acid and N-acetyl glucosamine units comprising the steps of:  
                 
(a) pre-preparation of the waste egg-shells for extraction of the embryonic low molecular weight aminoglycan compound of formula I using a polar organic solvent in water; (b) extraction of the low molecular weight aminoglycan compound of formula I as its soluble salt using an aqueous polar salt solution; (c) isolation of a purified low molecular weight aminoglycan compound of formula I by gel formation out of the aqueous salt mixture by using a polar organic solvent followed by filtration or centrifuging; (d) stabilization of the isolated aminoglycan extract by sequential introduction of organic oils into a semi-dried gel to form ordered sheets of aminoglycan compound of formula I with no visible cross-linking of the carbohydrate polymer.

FIELD

Embodiments of the invention relate to a method for simply andefficiently isolating and stabilizing ultra low molecular weightaminoglycans from waste egg shells.

BACKGROUND

Embodiments of the invention relate to processes for isolating,stabilizing and formulating low molecular weight aminoglycans from wasteegg shells. The aminolycan extract is useful for the preparation ofcosmetic creams with skin moisturizing and anti-wrinkle properties.

Nakano et al. (Poult Sci. (1991), Vol. 70(12), pp. 2524-8) have shownthat the chemical composition of glycosaminoglycan fractions from thecomb and wattle of single comb white Leghorn roosters consist of verylarge molecular weight glycosaminoglycans that have applications incartilage replacement therapy.

Balazs et al (U.S. Pat. No. 4,141,973) has described a process toisolated pure hyaluronic acid from animal tissue bearing molecularweights in the range of 1 MD to 6 MD useful as a replacement forsynovial fluids and vitreous humor.

Heaney et al. (Biochim Biophys Acta. (1976), Vol. 18;451(1), pp. 133-42)have shown that the organic part of the chicken's egg shell consists ofcollagen, proteins and polysaccharides which are probably present asglycoproteins and glycosaminoglycans. They further identified theorganic components by chromatography to yield glycosaminoglycans with aminimum molecular weight of 30,000 Daltons. Sedimentation velocityanalysis in a density gradient, showed that the polysaccharidescontained equimolar amounts of glucosamine (36.3% s/w) and glucuronicacid 35.6% w/w. Identification of the degradation products showed theglycosaminoglycan to be mainly hyaluronic acid.

Stahl et al (U.S. Pat. No. 6,537,795) have described a process toproduce and isolate aminoglycans from cultivated strains of streptococcifermentation. These aminoglycans are characterized by extreme highmolecular weights above 6 MD and are useful for cartilage replacementtherapy.

Related processes for isolations and purifications of glycosaminoglycansfrom other natural sources and animal tissues may also be found in U.S.Pat. No. 5,824,658, U.S. Pat. No. 6,660,853, U.S. Pat. No. 6,451,326.The references discussed within these patents are hereby incorporatedherein by reference.

SUMMARY

Embodiments of the invention provide novel processes for the isolationof low-molecular weight aminoglycan compounds of formula I from ahitherto unknown natural source of waste egg shells made up ofalternating glucuronic acid and N-acetyl glucosamine units

comprising:

(a) pre-preparation of the waste egg-shells for extraction of theembryonic low molecular weight aminoglycan compound of formula I using apolar organic solvent dissolved in water

(b) extraction of the low molecular weight aminoglycan compound offormula I as its soluble salt using an aqueous polar salt solution

(c) isolation of a purified low molecular weight aminoglycan compound offormula I by gel formation out of the aqueous salt mixture by using apolar organic solvent followed by filtration or centrifuging

(d) stabilization of the isolated aminoglycan extract by sequentialintroduction of organic oils into a semi-dried gel to form orderedsheets of aminoglycan compound of formula I with no visiblecross-linking of the carbohydrate polymer

Embodiments of the invention relate more particularly to step (b),wherein the aqueous polar salt solution can be the sodium, potassium,calcium or magnesium salts of citrate, glutamate, acetate,pyrrolidonecarbonate, tartrate, glycinate, sulfate, sulfite, nitrate,carbonate, oxalate to yield a solution containing aminoglycan compoundof formula I wherein M may be one or more instances of Na, Ca, K, Mg andn is an integer between 20 and 40, which is suitable for selectivegelation and isolation.

The processes described herein are novel methods to selectively andsimply yield a low-molecular weight aminoglycan compound of formula Ifrom waste egg shells. More specifically the processes of the invention,compared to the procedures for isolating aminoglycans disclosed in theprior art, are differentiated by;

-   -   a) identification of an novel hitherto unused source, egg shell        waste, which is otherwise difficult to dispose of and causes        significant negative impact to the environment,    -   b) contains very low concentrations of detrimental proteins and        nucleotides,    -   c) requires no expensive and inefficient separations of organic        and inorganic materials from the egg shell waste,    -   d) are simpler extractions involving mild reagents and solvents        materials and    -   e) requires no acetylation or other derivatization for example        using acetic anhydride and sulfuric acid as described in U.S.        Pat. No. 5,679,657 to achieve the desired viscosity and        threading properties needed for cosmetic applications.

The aminoglycan compound of formula I are of an unusually low molecularweight and are yet stabilized without derivatization to show excellentdermal penetration to reduce surface wrinkles in skin and exhibit anexcellent softening and moisturizing effect as well.

DETAILED DESCRIPTION

Egg shell waste produced from the egg processing industry is usuallywashed with solvents and treated to eliminate unpleasant smells beforebeing used as landfill. The calcium carbonate of the shells is onlyusable upon extensive separation and cleaning procedures which makes theprocess commercially uneconomical. There is no specific need topulverize the egg shells within a narrowly bound range either since theprocesses of the present invention are not dependent on the separationof the inner membrane from the egg shell as in the complex process andequipment described by MacNeil (U.S. Pat. No. 6,176,376) to arrive atpure calcium carbonate.

We have identified processes to selectively isolate valuable organiccompounds, specifically aminoglycan compound of formula I from crushedegg shells without the expensive separation of the organic and inorganiccomponents.

Crushed egg shells may be treated with warm water or warm 5% ethanolsolution and filtered to remove adhered organic wastes from the surfaceof the shells. The ratio of organic mass to calcium carbonate may bebetween 1% to 15% w/w. Greater ratios of organic mass would indicateunwashed egg mass present in the crushed egg shells which can lead tothe presence of detrimental protein and nucleotide products in theaminoglycan extract. It is noted that unlike other sources ofaminoglycans such as animal tissue and fermentation broths as known inthe prior art, the use of egg shell waste as shown herein is unique inthe absence of significant antigenic protein and nucleotide componentsin the extracted media leading to easier methods of extracting purifiedaminoglycan compound of formula I. The egg shells may be additionallypre-treated with ultraviolet light to destroy microbes which may bepresent even after liquid cleaning.

The next step comprises treatment of the above egg shell mass to ahighly selective extraction of the carbohydrate component in the form ofits water-soluble salt. The processes involve suspending the egg shellmass in 1:2 to 1:10 volume of solution containing 5% to 40% by weightcitrate, glutamate, acetate, pyrrolidonecarbonate, tartrate, glycinate,sulfate, sulfite, nitrate, carbonate and oxalate salts of sodium,potassium, calcium or magnesium or a combination of the above saltsolutions as needed. More specifically the mono-valent salts of organicacids are preferred. The suspension is held for 1 to 24 hours, morepreferably for 6 to 12 hours, with periodic vigorous shaking attemperatures ranging between 10 C and 35 C. The suspension issubsequently filtered or centrifuged to remove the aqueous solutioncontaining the appropriate salt of the aminoglycan compounds of formulaI. The egg shell mass thus separated shows a much looser binding of themembranes to the egg shell and hence may be more easily treated usingprocesses known in the art to separate the pure calcium carbonatecontaining egg shell from the organic residue.

The next step comprises the gel precipitation of the aminoglycan in itsappropriate salt form from the aqueous solution. The processes involvereducing the polarity of the aqueous solution and hence the solubilityof the aminoglycan by the sequential addition of any aqueous miscibleorganic solvent such as alcohols, acetone, dimethylformamide,N-methylpyrrolidinone or 1,4-dioxane. The organic solvent is added inlots with mild stirring and cooling to maintain the temperature of thereaction between 20 C to 25 C to yield a white gel formation suspendedin the aqueous layer. The solution is allowed to stand for 2 to 24 hoursuntil gelation is complete and subsequently filtered or centrifuged toyield a semi-dry extract of aminoglycan compound of formula I. It isimportant to not allow the extract to be completely dried since acertain amount of the aqueous phase is required during the stabilizationprocess carried out next.

The final step comprises the stabilization of the low-molecular weightaminoglycan compound of formula I by ordering the molecules in alipophilic environment to prevent cross-linking which is characteristicof non-acetylated and low-molecular weight aminoglycans as described inthe prior art. The process involves a sequential addition of two oilswhose total weight ratio to the aminoglycan extract is between 1:0.5 and1:3 of aminoglycan to oils and wherein the individual oil ratios betweenthe two oils are 3:1 to 8:1. The first oils should be more hydrophobicin nature and may be oils found typically in plant nuts. Specificallyalmond and jojoba oils are more preferred as the first oil. The secondoil component should be more hydrophilic in nature and may be oilstypical isolated from herbs and spices from the vegetative parts ofplants. Specifically sage, rosemary and lavender oils are more preferredas the second oils.

The molecular weight of aminoglycan compound of formula I thus isolatedis difficult to measure directly and hence I have relied on the measureof intrinsic viscosity to determine the molecular weight. The intrinsicviscosities of various solutions containing aminoglycan compound offormula I were found to lie between 4 cm3/gm and 7 cm3/gm and whenplotted against standard solutions of hyaluronic acid salts (Mol. Wt.approximately 1.2 MD) led to the assignment of a unique natural ultralow molecular weight for aminoglycan compound of formula I between therange of 15,000 Daltons and 28,000 Daltons. An ultra low molecularweight aminoglycan compound of formula I from a natural source has notbeen previously described in the prior art (for example as summarized byBalazs et al. in U.S. Pat. No. 4,582,865).

Experimental Results

EXAMPLE 1

500 grams of pre-treated egg shell waste with approximately 10% organiccontent was added to an open mouth glass container with a screw top. Tothis was added 750 ml of a 5% aqueous solution of sodium citrate and thecontainer sealed and placed on a shaker for 24 hours at moderate speeds.After 24 hours the entire mixture was transferred into a filter funneland the solid egg shell waste was separated from the aqueous suspension.The solids were washed 1× with 250 ml of 5% aqueous solution of sodiumcitrate and the combined aqueous layers were washed once with 250 ml ofmethylene chloride to remove potential proteinaceous matter and theaqueous layer then transferred to a 2L beaker. The beaker was placed ina cold water bath and a slow addition of absolute methanol was initiatedwith slow stirring. After about 200 ml of methanol addition wascompleted, a cloudy white precipitate began to form and the stirring wasstopped. An equal additional quantity of methanol was added slowly andthe beaker allowed to stand for 12 hours to make sure gelation wascomplete. The entire mass was transferred to a filter funnel andfiltered to give a cream colored gel of aminoglycan compound of formulaI. The precipitate was dried until a moisture content of 5-7% wasmeasured. The final weight of the gel of aminoglycan compound of formulaI was 42 grams.

EXAMPLE 2

The gel material containing aminoglycan compound of formula I fromExample 1 was mixed with 4 grams of jojoba oil at 15-20 C and stirredvigorously for 20 minutes. The resultant gel was warmed to 25 C andallowed to gently stir for 1 hour. To this mass was added 1 gram of sageoil and the resultant gel was further stirred gently for 10 minutes. Thegel is then allowed to slowly cool to 10 C over 4 hours whereby theaminoglycan compound of formula I is ordered into a secondary structurethat is stable in the absence of circulating air at room temperature forat least 3 months.

EXAMPLE 3

The above example 1 was repeated with a 10% aqueous solution ofpotassium tartrate to yield 46 grams of the gel of aminoglycan compoundsof formula I.

EXAMPLE 4

The above example 1 was repeated with a 20% solution of sodium acetateto yield 43 grams of the gel of aminoglycan compound of formula I.

EXAMPLE 5

The above example 4 was repeated with a final gelation using ethanolinstead of methanol to yield 47 grams of the gel of aminoglycan compoundof formula I.

EXAMPLE 6

The above example 5 was repeated with a final gelation using acetoneinstead of ethanol to yield 41 grams of the gel of aminoglycan compoundof formula I.

EXAMPLE 7

The above example 1 was repeated with a 10% solution of sodium carbonateto yield 24 grams of the gel of aminoglycan compound of formula I.

EXAMPLE 8

The above example 1 was repeated with a 25% solution of calciumcarbonate to yield 14 grams of the gel of aminoglycan compound offormula I.

EXAMPLE 9

10 grams of the above stabilized gel made as per the procedure shown inExample 2 is added to 50 ml of distilled water containing 3 ml ofglycerin and stirred to a uniform suspension. To this suspension isadded a melt consisting of 10 grams of emulsifying wax, 10 grams ofparaffin wax, 4 grams of white beeswax and 13 grams of a mix ofcosmetically useful plant oils such as almond, lavender, sandalwood andwalnut and the mixture stirred vigorously to give a uniform cream withexcellent physical characteristics and anti-wrinkle properties.

With respect to the above isolated and stabilized gels of aminoglycancompound of formula I the following analytical and usefulness tests wereconducted.

Absence of Chondroitin Sulfate

It is known in the prior art that all commercial sources of aminoglycansare usually closely associated with other tissue components such asChondroitin sulfate (Arkins and Sheehan, Structure of Hyaluronic Acid,Nature New Biol 235, 253, 1972 and Bettelheim and Philpott, ElectronMicroscopic Studies of Hyaluronic Acid—Protein Gels, Biochim BiophysActa 34, 124, 1959). The gel extract isolated as per the methodsdescribed above contains less than 2% Chondroitin sulfate probably dueto the low association possible with the extra small size of theaminoglycan compound of formula 1 isolated herein.

Absence of Proteins

Since proteins are potentially antigenic, it is essential for cosmeticformulations to isolate any aminoglycan gel essentially free ofproteins. The gel extract from Example 1 was subjected to the highlysensitive colorimetric test for detecting the presence of proteinsdescribed by Lowry et al. (J. Biol. Chem., 193, 265-275, 1951) Nopositive result was obtained indicating the presence of proteins to beless than 0.1% by weight.

The absence of any appreciable protein concentration is a distinctdifference from other glycolaminoglycan compounds isolated from othernatural sources such as Rooster Comb and fermentation broths. It hasbeen reported (Kludas, U.S. Pat. No. 5,055,298) that these aminoglycansare usually covalently linked with proteins to form proteoglycans.Clinically relevant removal of all of these proteins, which are notcomponents of human skin, has proved to be difficult and not easilyaccomplished. The presence of these proteins in various otheraminoglycan extracts has been identified as a cause of significantinflammatory responses on skin surfaces, making their use in cosmeticformulations challenging.

Absence of Nucleotides

Ultraviolet spectroscopy has been used to show the absence ofpotentially antigenic DNA and RNA nucleotides in the aminoglycancompound of formula I extracted herein. A 1% solution of the aminoglycanextract from Example 1 in 10% sodium chloride solution was prepared.This solution was subjected to ultraviolet spectroscopy at 257nanometers to measure the level of nucleotides in the solution. Theabsence of any absorption at this wavelength was taken as a measure ofthe absence of nucleotides in the aminoglycan extract from Example 1.

Viscosity

A small sample of the gel was freeze-dried to give a white solid with athread like structure which slowly dissolved in water. A solution of 1gram of the powder was made up in 1000 ml of a phosphate buffer at pH 7.Viscosity was determined with an Ostwald viscosimeter at a temperatureof 25 C. The relative viscosity of the solution was measured as 0.76 to0.80. When compared to aminoglycans of known higher molecular weightthis viscosity measurement leads to molecular weights for aminoglycancompound of formula I between 15 kD and 28 kD.

Glucosamine Presence

The presence of glucosamine in the aminoglycan compound of formula I wasdetermined by the method of Elson and Morgan (Biochem J, Vol. 27,(1933), p. 1894,) on material that had been hydrolyzed for 6 hours with5N hydrochloric acid at 100 C and evaporated to dryness. The glucosaminecontent of the aminoglycan compound of formula I was between 38% and 41%which matches the expected calculated value.

Thread Forming Ability

It is well documented in the prior art that the higher the threadforming ability the more moisturizing is the effect of the aminoglycan.Many derivatives of high and medium molecular weight aminoglycans suchas acetylation and co-polymerization (U.S. Pat. No. 5,679,657) have beenused to increase the intrinsic threading value of aminoglycans isolatedfrom animal and bacterial sources. It is unexpectedly observed that theultra low molecular weight aminoglycan compound of formula I isolatedherein shows a remarkably high thread forming ability and may accountfor part of the high anti-wrinkle effects observed. In a humiditychamber at a temperature of 25 C and relative humidity of 50%, 1 cm of aglass rod was immersed in a 1% aqueous solution of aminoglycan extractfrom Example 1 and the thread length obtained upon lowering the beakerat a velocity of 10 cm/min was observed. The thread length of theaminoglycan of this invention was observed to be between 2.8 cm and 3.5cm which is considerably longer than the 0.8 cm to 1.3 cm observed forcommercially available sodium hyaluronate and even better than thelengths observed for derivatized aminoglycans.

Anti-Wrinkle Properties

The anti-wrinkle properties of the cream produced as per the methoddescribed in Example 9 was tested using a 3D imaging system to measuredepths of surface wrinkles. The method described by S. Jaspers et al,(“Microtopometry Measurement of Human Skin in vivo by a new DigitalOptical Projection System”, Preprints 5th Congress of the InternationalSociety for Skin Imaging, Wien 1997) was used to show a 25% to 38%reduction in wrinkle depth after 4 weeks of daily use.

1. A process for the isolation of low-molecular weight aminoglycancompounds of formula I from waste egg shells,

wherein M may be one or more instances of Na, Ca, K, Mg and n is aninteger between 20 and 40; said Process comprising the steps of: (a)pre-preparation of the waste egg-shells for extraction of the embryoniclow molecular weight aminoglycan compound of formula I using a polarorganic solvent in water, wherein the pretreated egg shells are mixedthoroughly with the polar organic solvent in water at temperaturesbetween 25° C. and 40° C. for 1 hour to 4 hours, followed by decantationof the supernatant and the eggshells carried forward for extraction; (b)extraction of the low molecular weight aminoglycan compound of formula Ias its water soluble salt using an aqueous polar salt solution, whereinthe eggshells from step (a) are vigorously shaken with the aqueous polarsalt solution at 25° C. to 40° C. for 6 to 24 hours, followed bydecantation, filtration or centrifugation to collect the aqueous layercontaining the dissolved aminoglycan compound of formula I; (c)isolation of a purified low molecular weight aminoglycan compound offormula I by gel formation out of the aqueous salt mixture by using apolar organic solvent, wherein the solution from step (b) is subjectedto sequential step wise addition of the polar organic solvent in anamount between 75% and 150% volume/volume of the polar organic solventbetween 10° C. and 20° C. in 1 hour to 2 hours and the gel formed isallowed to stand for 4 hours to 12 hours to complete precipitation,followed by decantation, filtration or centrifugation to isolate semidried aminoglycan compound of formula I containing between 4% to 8% ofmoisture; (d) stabilization of the isolated aminoglycan compound offormula I from step (c) by sequential introduction of organic oils intothe semi-dried gel to form the aminoglycan compound of formula I.
 2. Theprocess as claimed in claim 1, wherein the polar organic solvent used instep (a) is selected from the group consisting of an alcohol, acetone,methylethylketone or 1,4-dioxane.
 3. The process as claimed in claim 1,wherein the said aqueous polar salt solution used in step (b) is anorganic acid salt selected from the group consisting of sodium,potassium, calcium or magnesium salt of citrate, glutamate, acetate,pyrrolidone carbonate, tartrate, glycinate, sulfate sulfite, nitrate,carbonate or oxalate.
 4. The process as claimed in claim 1, wherein thepolar organic solvent used in step (c) is a lower alcohol selected frommethanol, ethanol, propanol or butanol, or an organic ether selectedfrom diethylether, tetrahydrofuran, methylal or ethylal.
 5. The processas claimed in claim 1, wherein the organic oils used in step (d) are theoils obtained from plant sources.
 6. The process as claimed in claim 5,wherein the said organic oil is selected from jojoba, almond, sage,rosemary, lavender, sandalwood or aloe oil.